Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation

Abstract

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.

Figure 1

Structure and biochemical characterisation of PAD4 inhibitors. a) Summary of biochemical potency data from binding and functional assays for PAD4 inhibitors and control compound GSK106. The FP binding assay was run at a range of calcium concentrations to assess dependency. Replicate numbers are indicated in parentheses (ND = not determined). b) BAEE displacement of 10 nM GSK215 binding from PAD4 in the absence of calcium; the IC₅₀ for BAEE was calculated as 12.5 mM. Analogous experiments in the presence of added calcium were difficult to configure and interpret due to enhanced catalytic activity and hence turnover of BAEE.

Figure 2

GSK199 binds to a re-ordered PAD4 active site. a) 3.29 Å PAD4/GSK199 complex showing hydrogen bonding interactions as dotted red lines. PAD4 is shown in cyan except for residues 633–645 (yellow), which form a hydrophobic lid with F634 and V643 packing ‘on top’ of GSK199 in this view. b) Overlay of calcium-free PAD4 structure (1WD8 – dark blue, with disordered residues 633–645 indicated by dashed blue line) with the structure of PAD4/GSK199. c) Overlay of calcium-free PAD4 structure (1WD8 – dark blue, with disordered residues 633–645 indicated by dashed blue line) with PAD4/benzoyl-L-arginine amide (BAA – green, PDB:1WDA) which has the substrate analogue inhibitor (BAA – green stick format) bound at the active site (C645A mutant). (d) Overlay of the PAD4/GSK199 and PAD4/BAA structures.

Figure 3

Inhibition of mouse NETs. a) Image of control unstimulated neutrophils. b) Image of calcium ionophore-stimulated neutrophils. c) and d) Representative images of stimulated neutrophils in the presence of 10 µM GSK484 or 10 µM GSK106, respectively. All images are representative of n=5–10. Immunostaining for citrullinated histone H3 (green) was performed with DNA counterstained with Hoechst 33342 (blue). White arrows indicate NETs identified as H3Cit⁺ cells with spread nuclear morphology. Scale bar = 20 µm. e) Quantification of hypercitrullinated H3-stained neutrophils following ionomycin stimulation. Results are expressed as mean ± SEM and are representative of n=11 (unstimulated), n=13 (ionomycin) or n=5–9 for treatment with GSK199, GSK484 or GSK106. f) Quantification of NET-forming neutrophils following ionomycin stimulation. NETs were identified as H3Cit⁺ cells with spread nuclear morphology as visualised by Hoechst 33342 stain. Results are representative of n=10 (unstimulated and ionomycin treatments) or n=5–9 for treatment with GSK199, GSK484 or GSK106. *p<0.05, *** p<0.001. Effects of the inhibitors on neutrophil viability were ruled out (Supplementary Fig 16a).